The present invention relates to the use of primers in polymerase chain reaction assays for the detection of stone fruit and nut, in particular almond pathogens Colletotrichum acutatum, Alternaria spp., and Cladosporium carpophilum. The use of these primers enables the detection of specific isolates of fungal pathogens and the monitoring of disease development in plant populations. The present invention also relates to novel extraction buffer solutions, methods of extracting DNA from tissue, and methods of performing PCR analysis on DNA extracted from tissue.
Diseases in plants cause considerable crop loss from year to year resulting both in economic deprivation to farmers and, in many parts of the world, to shortfalls in the nutritional provision for local populations. The widespread use of fungicides has provided considerable security against plant pathogen attack. However, despite $1 billion worth of expenditure on fungicides, worldwide crop losses amounted to approximately 10% of crop value in 1981 (James, 1981; Seed Sci. and Technol. 9: 679-685).
The severity of the destructive process of disease depends on the aggressiveness of the pathogen and the response of the host. One aim of most plant breeding programs is to increase the resistance of host plants to disease. Typically, different races of pathogens interact with different varieties of the same crop species differentially, and many sources of host resistance only protect against specific pathogen races. Furthermore, some pathogen races show early signs of disease symptoms, but cause little damage to the crop. Jones and Clifford (1983; Cereal Diseases, John Wiley) report that virulent forms of the pathogen are expected to emerge in the pathogen population in response to the introduction of resistance into host cultivars and that it is therefore necessary to monitor pathogen populations. In addition, there are several documented cases of the evolution of fungal strains that are resistant to particular fungicides. As early as 1981, Fletcher and Wolfe (1981; Proc. 1981 Brit. Crop Prot. Conf.) contended that 24% of the powdery mildew populations from spring barley and 53% from winter barley showed considerable variation in response to the fungicide triadimenol and that the distribution of these populations varied between varieties, with the most susceptible variety also giving the highest incidence of less susceptible types. Similar variation in the sensitivity of fungi to fungicides has been documented for wheat mildew (also to triadimenol), Botrytis (to benomyl), Pyrenophora (to organomercury), Pseudocercosporella (to MBC-type fungicides) and Mycosphaerella fijiensis to triazoles to mention just a few (Jones and Clifford; Cereal Diseases, John Wiley, 1983).
Commercial almond growers are faced with a number of fungi that infect their crops in diverse ways. The impact of these pathogens on tree growth depends on a number of factors, and the cause is not immediately apparent from the symptoms a diseased tree may present. Vascular pathogens invade and plug the xylem vessels, thereby halting movement of water and nutrients up from roots (Integrated Pest Management for Almonds, U. California Division of Agriculture and Natural resources publication 3308 (1985)). Symptoms often reflect a partial or complete cut-off from food or water as vascular tissues are closed. Thus, symptoms of crown and root rots of almond, as well as wilts caused by pathogens infecting leaf and branch tissues, may appear very similar to one another; and similar to the consequences of environmental conditions such as the availability of nutrients and water and factors affecting their uptake.
Some pathogens cause infections that are limited to branches, foliage, and fruit. Several of the fungi and bacteria that cause disease in almonds have been found to infect orchards during the fall months and over-winter in host tissues. During the spring, they produce localized lesions or larger infections that become evident after environmental conditions such as a recent rain or irrigation promote the growth of the pathogens.
The same pathogens that cause economic problems in almond orchard management also affect other crops. For instance, Cladosporium carpophilum has a host range that extends over stone fruits. For example, in addition to the scabs it causes in almond crops, C. carpophilum also causes scabs in peaches, nectarines, apricots, plums, and cherries (Compendium of Stone Fruit Diseases, Ogawa, J. M.; Zehr, E. I.; Bird, G. W.; Ritchie, D. F.; Uriu, K.; Uyemoto, J. K. eds., p. 11 (1995 APS Press, St. Paul, Minn.)).
Alternaria spp. has been documented as causing infection on many economically important crops. To date, it has been reported to cause alternaria blight of peas, and alternaria leaf spots have been reported on peanuts, almonds, corn, cotton, and in all soybean growing areas of the world (Compendium of Soybean Diseases, 4th Ed. Hartman, G. L.; Sinclair, J. B.; Rupe, J. C. eds., pp. 12-13 (1999, APS Press, St. Paul, Minn.)); (Compendium of Cotton Diseases, Watkins, G. M. ed., p. 28 (1981, APS Press, St. Paul, Minn.); Compendium of Pea Diseases, Hagedorn, D. J., ed., p. 15 (1984, APS Press, St. Paul, Minn.)); (Compendium of Peanut Diseases, Porter, D. M.; Smith, D. H.; Rodriguez-Kabana, R. eds. Pp. 13 (1984, APS Press, St. Paul, Minn.); (Compendium of Stone Fruit Diseases, Ogawa, J. M.; Zehr, E. I.; Bird, G. W.; Ritchie, D. F.; Uriu, K.; Uyemoto, J. K. eds., p. 11 (1995 APS Press, St. Paul, Minn.)); and Compendium of Corn Diseases, 3rd Ed. White, D. G., ed., p. 25 (1999, APS Press, St. Paul, Minn.)). Alternaria spp. is responsible for widespread leaf and pod spots on beans grown in Brazil, Cananda, Costa Rica, Colombia, Chile, East Africa, England, Mexico, the United States, and Venezuela (Compendium of Bean Diseases, Hall, R. ed., p. 14 (1991, APS Press, St. Paul, Minn.)). Alternaria spp. also form lesions on potatoes and the leaves of other solanaceous crops; (Compendium of Potato Diseases, Hooker, W. J. ed., p. 44 (1981, APS Press, St. Paul, Minn.)) and appear as secondary infections on sugar beet leaves (Compendium of Beet Diseases and Insects, Whitney, E. D., Duffus, J. E. eds., p. 11 (1991 APS Press, St. Paul, Minn.)). It causes grain molds in sorghum (Compendium of Sorghum Diseases, Frederiksen, R. A. ed., p. 36 (1986 APS Press, St. Paul, Minn.)) and is one cause of black mold rot of tomato (Compendium of Tomato Diseases, Jones, J. B.; Jones, J. P.; Stall, R. E.; Zitter, T. A., eds., p. 46 (1991 APS Press, St. Paul, Minn.)). Alternaria spp. also causes fruit spot of papaya, a major disease in orchards located in dry areas and in mangos (Compendium of Tropical Fruit Diseases, Ploetz, R. C.; Zentmyer, G. A.; Nishijima, W. T.; Rohrbach, K. G.; Ohr, H. D., eds., pp. 34 and 58 (1994 APS Press, St. Paul, Minn.)).
Colletotrichum acutatum has been reported to infect a large number of fruit crops including avocado, strawberry, almond, apple, and peach. C. acutatum causes post-harvest fruit diseases in avocado and mango. C. acutatum is also known to cause both post-bloom fruit drop and key lime anthracnose in citrus crops (see Freeman, S. et al., 1998, Plant Disease Vol. 82, No. 6, pp. 596-605).
In view of the above, there is a real need for the development of technology that will allow the identification of specific races of pathogenic fungi early in the infection process. By identifying the specific race of a pathogen before disease symptoms become evident in the crop stand, the agriculturist can assess the likely effects of further development of the pathogen in the crop variety in which it has been identified and can choose an appropriate fungicide if such application is deemed necessary.
The present invention is drawn to methods of identification of different pathotypes of plant pathogenic fungi. The invention provides Internal Transcribed Spacer (ITS) DNA sequences that show variability between different fungal pathotypes. Such DNA sequences are useful in the method of the invention as they can be used to derive primers for use in polymerase chain reaction (PCR)-based diagnostic assays. These primers generate unique fragments in PCR reactions in which the DNA template is provided by specific fungal pathotypes and can thus be used to identify the presence or absence of specific pathotypes in host plant material before the onset of disease symptoms.
In a preferred embodiment, the invention provides ITS-derived diagnostic primers for the detection of Colletotrichum acutatum, Alternaria spp., and Cladosporium carpophilum. 
This invention provides the possibility of assessing potential damage in a specific crop variety/pathogen strain relationship and of utilizing judiciously the diverse armory of fungicides that is available. Furthermore, the invention can be used to provide detailed information on the development and spread of specific pathogen races over extended geographical areas. The invention provides a method of detection that is especially suitable for diseases with a long latent phase.
Kits useful in the practice of the invention are also provided. The kits find particular use in the identification of Colletotrichum acutatum, Alternaria spp., and Cladosporium carpophilum. 
The invention also provides methods for preparing an extract of DNA from tissue using novel DNA extraction buffer. The invention further provides for methods for performing PCR analysis on DNA extracted from tissue using the novel DNA extraction buffer and methods of preparing an extract of DNA.
SEQ-ID-NO: 1 Oligonucleotide Primer ITS1
SEQ-ID-NO: 2 Oligonucleotide Primer ITS2
SEQ-ID-NO: 3 Oligonucleotide Primer ITS3
SEQ-ID-NO: 4 Oligonucleotide Primer ITS4
SEQ-ID-NO: 5 Oligonucleotide Primer FORWARD
SEQ-ID-NO: 6 Oligonucleotide Primer REVERSE
SEQ-ID-NO: 7 Oligonucleotide Primer CaINT-1
SEQ-ID-NO: 8 Oligonucleotide Primer CaINT-2
SEQ-ID-NO: 9 Oligonucleotide Primer CaInt2
SEQ-ID-NO: 10 Oligonucleotide Primer JB677
SEQ-ID-NO: 11 Oligonucleotide Primer JB678
SEQ-ID-NO: 12 Oligonucleotide Primer JB679
SEQ-ID-NO: 13 Oligonucleotide Primer Alal-1
SEQ-ID-NO: 14 Oligonucleotide Primer Alal-2
SEQ-ID-NO: 15 Oligonucleotide Primer Alal-3
SEQ-ID-NO: 16 Oligonucleotide Primer Alal-4
SEQ-ID-NO: 17 Oligonucleotide Primer Alal-5
SEQ-ID-NO: 18 Oligonucleotide Primer Alal-6
SEQ-ID-NO: 19 Oligonucleotide Primer Alt1
SEQ-ID-NO: 20 Oligonucleotide Primer Alt2
SEQ-ID-NO: 21 Oligonucleotide Primer Vcarp1
SEQ-ID-NO: 22 Oligonucleotide Primer Vcarp2
SEQ-ID-NO: 23 Oligonucleotide Primer Vcarp3
SEQ-ID-NO: 24 Oligonucleotide Primer Vcarp4
SEQ-ID-NO: 25 Oligonucleotide Primer Vcarp5
SEQ-ID-NO: 26 Oligonucleotide Primer Vcarp6
SEQ-ID-NO: 27 Oligonucleotide Primer Vcarp7
SEQ-ID-NO: 28 Oligonucleotide Primer JB677.1
SEQ-ID-NO: 29 Oligonucleotide Primer JB677.2
SEQ-ID-NO: 30 Oligonucleotide Primer JB677.3
The present invention provides unique DNA sequences that are useful in identifying different pathotypes of plant pathogenic fungi. Particularly, the DNA sequences can be used as primers in PCR-based analysis for the identification of fungal pathotypes. The DNA sequences of the invention include the Internal Transcribed Spacer (ITS) sequences of the ribosomal RNA gene regions of particular fungal pathogens as well as primers derived from these regions that are capable of identifying the particular pathogen. ITS DNA sequences from different pathotypes within a pathogen species or genus, which vary between the different members of the species or genus, can be used to identify those specific members.
Biomedical researchers have used PCR-based techniques for some time and with moderate success to detect pathogens in infected animal tissues. Only recently, however, has this technique been applied to detect plant pathogens. The presence of Gaumannomyces graminis in infected wheat has been detected using PCR of sequences specific to the pathogen mitochondrial genome (Schlesser et al., 1991; Applied and Environ. Microbiol. 57: 553-556), and random amplified polymorphic DNA (i.e. RAPD) markers were able to distinguish numerous races of Gremmeniella abietina, the causal agent of scleroderris canker in conifers. U.S. Pat. No. 5,585,238 (herein incorporated by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of Septoria, Pseudocercosporella, and Mycosphaerella and their use in the identification of these fungal isolates using PCR-based techniques. In addition, U.S. Pat. No. 5,955,274 (herein incorporated by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of Fusarium and their use in the identification of these fungal isolates using PCR-based techniques. Furthermore, U.S. Pat. No. 5,800,997 (herein incorporated by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of Cercospora, Helminthosporium, Kabatiella, and Puccinia and their use in the identification of these fungal isolates using PCR-based techniques.
Ribosomal genes are suitable for use as molecular probe targets because of their high copy number. Despite the high conservation between mature rRNA sequences, the non-transcribed and transcribed spacer sequences are usually poorly conserved and are thus suitable as target sequences for the detection of recent evolutionary divergence. Fungal rRNA genes are organized in units, each of which encodes three mature subunits of 18S (small subunit), 5.8S, and 28S (large subunit). These subunits are separated by two Internal Transcribed Spacers, ITS1 and ITS2, of around 300 bp (White et al., 1990; In: PCR Protocols; Eds.: Innes et al.; pages 315-322). In addition, the transcriptional units are separated by non-transcribed spacer sequences (NTSs). ITS and NTS sequences are particularly suitable for the detection of specific pathotypes of different fungal pathogens.
The DNA sequences of the invention are from the Internal Transcribed Spacer sequences of the ribosomal RNA gene region of different plant pathogens. The ITS DNA sequences from different pathotypes within a pathogen species or genus vary among the different members of the species or genus. Once having determined the ITS sequences of a pathogen, these sequences can be aligned with other ITS sequences. In this manner, primers can be derived from the ITS sequences. That is, primers can be designed based on regions within the ITS sequences that contain the greatest differences in sequence among the fungal pathotypes. These sequences and primers based on these sequences can be used to identify specific pathogens.
Sequences of representative oligonucleotide primers derived from ITS sequences are disclosed in SEQ-ID-NOs: 1-4 and 7-30. The sequences find use in the PCR-based identification of the pathogens of interest. In a preferred embodiment, the primer comprises a nucleotide sequence of SEQ ID NOs: 10-30.
The present invention also provides pairs of oligonucleotide primers. In one embodiment, the pair comprises or consists of at least one primer of SEQ ID NO: 1-4 or 7-30. In a preferred embodiment, the pair consists of at least one primer of SEQ ID NO: 10-30.
In yet other embodiments, a pair of oligonucleotide primers, is selected from the group consisting of: SEQ ID NO:30 and SEQ ID NO:4; SEQ ID NO:27 and SEQ ID NO:4; SEQ ID NO: 16 and SEQ ID NO: 12; SEQ ID NO: 16 and SEQ ID NO: 18; SEQ ID NO: 17 and SEQ ID NO: 12; SEQ ID NO: 1 and SEQ ID NO:27; SEQ ID NO:24 and SEQ ID NO:25; and SEQ ID NO:21 and SEQ ID NO:4.
In preferred embodiments, the pair of oligonucleotide primers consists of SEQ ID NO:16 and SEQ ID NO:12; or SEQ ID NO:16 and 18; or SEQ ID NO:17 and SEQ ID NO: 12; or SEQ ID NO:24 and SEQ ID NO:25.
Methods for the use of the primer sequences of the invention in PCR analysis are well known in the art. For example, see U.S. Pat. Nos. 4,683,195 and 4,683,202, as well as Schiesser et al. (1991) Applied and Environ. Microbiol. 57:553-556. See also, Nazar et al. (1991; Physiol. and Molec. Plant Pathol. 39:1-11), which used PCR amplification to exploit differences in the ITS regions of Verticillium albo-atrum and Verticillium dahliae and therefore distinguish between the two species; and Johanson and Jeger (1993; Mycol. Res. 97: 670-674), who used similar techniques to distinguish the banana pathogens Mycosphaerella fijiensis and Mycospharella musicola. 
The target DNA sequences of the invention can be cloned from fungal pathogens by methods known in the art. In general, the methods for the isolation of DNA from fungal isolates are known. See, Raeder and Broda (1985) Letters in Applied Microbiology 2:17-20; Lee et al. (1990) Fungal Genetics Newsletter 35:23-24; and Lee and Taylor (1990) In: PCR Protocols: A Guide to Methods and Applications, Innes et al (Eds.); pages 282-287. The ITS rDNA sequences are compared within each pathogen group to locate divergences that might be useful to test in PCR to distinguish the different species and/or strains. From the identification of divergences, numerous primers are synthesized and tested in PCR-amplification. Templates used for PCR-amplification testing are firstly purified pathogen DNA, and subsequently DNA isolated from infected host plant tissue. Thus, it is possible to identify pairs of primers that are diagnostic, i.e. that identify one particular pathogen species or strain but not another species or strain of the same pathogen. Primers are also designed to regions highly conserved among the species to develop genus-specific primers as well as primers that will identify any of several fungal pathogens that cause a particular disease. For example, primers are developed to differentiate Colletotrichum acutatum, Alternaria spp., and Cladosporium carpophilum. 
Preferred primer combinations are able to distinguish between the different species or Ad strains in infected host tissue, i.e. host tissue that is infected with a specific pathogen genus, species or strain. This invention provides numerous primer combinations that distinguish Colletotrichum acutatum, Alternaria spp., and Cladosporium carpophilum. The primers of the invention are designed based on sequence differences among the ITS rDNA regions. A minimum of one base pair difference between sequences can permit design of a discriminatory primer. Primers designed to a specific fungal DNA sequence can be used in combination with a primer made to a conserved sequence region flanking the region containing divergences to amplify species-specific PCR fragments. In general, primers should have a theoretical melting temperature between about 60 to about 70 degree xc2x0 C. to achieve good sensitivity and should be void of significant secondary structure and 3xe2x80x2 overlaps between primer combinations. In preferred embodiments, primers are anywhere from approximately 5-30 nucleotide bases long.
The present invention provides, a method for the detection of a fungal pathogen, comprising the steps of:
(a) isolating DNA from a plant tissue infected with a pathogen;
(b) subjecting said DNA to polymerase chain reaction amplification using at least one primer according to SEQ ID NO: 1-4 and 7-30; and
(c) detecting said fungal pathogen by visualizing the product or products of said polymerase chain reaction amplification.
In a preferred embodiment, the primer is at least one according to SEQ ID NO: 10-30.
In another embodiment, the method for detection is used when the fungal pathogen is Colletotrichum acutatum, Alternaria spp., and Cladosporium carpophilum. 
In another embodiment, the invention provides a method for the detection of a fungal pathogen, comprising the steps of:
(a) isolating DNA from a plant tissue infected with said fungal pathogen;
(b) amplifying a part of the Internal Transcribed Spacer sequence of said fungal pathogen using said DNA as a template in a polymerase chain reaction with a pair of primers wherein the pair comprises or consists of at least one primer of SEQ ID NO: 1-4 or 7-10. In a preferred embodiment, the pair consists of at least one primer of SEQ ID NO: 10-30; and
(c) detecting said fungal pathogen by visualizing the amplified part of the Internal Transcribed Spacer sequence.
In a preferred embodiment, the method detects a fungal pathogen, wherein said fungal pathogen is Colletotrichum acutatum, Alternaria spp., and Cladosporium carpophilum. In yet other embodiments, the method of detection uses a pair of primers, wherein said pair of primers is selected from the group consisting of: SEQ ID NO: 30 and SEQ ID NO:4; SEQ ID NO:27 and SEQ ID NO:4; SEQ ID NO:16 and SEQ ID NO:12; SEQ ID NO:16 and SEQ ID NO: 18; SEQ ID NO: 17 and SEQ ID NO: 12; SEQ ID NO: 1 and SEQ ID NO:27; SEQ ID NO:24 and SEQ ID NO:25; and SEQ ID NO:21 and SEQ ID NO:4.
In preferred embodiments, the pair of oligonucleotide primers consists of SEQ ID NO: 16 and SEQ ID NO: 12; or SEQ ID NO: 16 and 18; or SEQ ID NO: 17 and SEQ ID NO: 12; or SEQ ID NO:24 and SEQ ID NO:25.
The present invention also provides a method for performing PCR analysis on DNA extracted from tissue, comprising the steps of:
(a) taking a plurality of random tissue samples from an organism population;
(b) adding the extraction buffer described in Example 2, to the tissue samples;
(c) macerating the tissue samples and extraction buffer to form an extract;
(d) removing the extract from the macerated tissue and buffer; and
(e) performing PCR analysis on the extract.
In another embodiment, the method further comprises the step of boiling the extract after removing it from the macerated tissue and buffer.
In yet another embodiment, the method further comprises the step of diluting the extract.
In a preferred embodiment, the method uses an organism population that is a plant population. In more preferred embodiments, the method uses tissue samples selected from leaves, stems, roots, blossoms, immature flowers, peduncles, hulls, fruits, immature fruits, or woody tissue.
In another preferred embodiment, the method uses the extraction buffer comprising: 100 mM Tris, pH 8.0; 1.4 M NaCl; 20 mM EDTA; 2% w/v CTAB; 2% w/v PVP and 0.1% w/v ascorbic acid.
The present invention lends itself readily to the preparation of xe2x80x9ckitsxe2x80x9d containing the elements necessary to carry out the process. Such a kit may comprise a carrier being compartmentalized to receive in close confinement therein one or more container, such as tubes or vials. One of the containers may contain unlabeled or detectably labeled DNA primers. The labeled DNA primers may be present in lyophilized form or in an appropriate buffer as necessary. One or more containers may contain one or more enzymes or reagents to be utilized in PCR reactions. These enzymes may be present by themselves or in admixtures, in lyophilized form or in appropriate buffers.
Finally, the kit may contain all of the additional elements necessary to carry out the technique of the invention, such as buffers, extraction reagents, enzymes, pipettes, plates, nucleic acids, nucleoside triphosphates, filter paper, gel materials, transfer materials, autoradiography supplies, and the like.
In an embodiment of the invention, the diagnostic kit used in detecting a fungal pathogen, comprises a primer of the present invention as described above.
In yet another embodiment, the diagnostic kit used in detecting a fungal pathogen, comprises a pair of primers of the present invention as described above. The primers, methods and kits of the present invention are useful for detecting the presence of fungal pathogens in any plants or plant parts that are infected by fungal pathogens. In particular, the present invention is useful for detection of Colletotrichum acutatum, Alternaria spp., and Cladosporium carpophilum. Examples of plants or plant tissues or plant parts infected by these pathogens include, but are not limited to, stone fruits, nuts, solanaceous plants such as tomato and potato, peanuts, corn, sorghum, peas, papaya, avacado, apples, and sugarbeets. Examples of stone fruits include, but are not limited to peaches, nectarines, cherries, apricots and plums. Examples of nut crops include, but are not limited to peanuts, almonds, walnuts, cashews, hazelnuts, brazil nuts, etc.
The examples below show typical experimental protocols that can be used in the selection of suitable primer sequences, the testing of primers for selective and diagnostic efficacy, and the use of such primers for disease and fungal isolate detection. Such examples are provided by way of illustration and not by way of limitation.